The G protein-coupled receptor (GPCR) beta-2-adrenergic receptor (Beta2AR) plays an important role in regulating airway smooth muscle (ASM) contractile state and other important ASM functions including synthesis of molecules that modulate airway inflammation. Consequently, beta-sympathomimetic drugs are the most widely used agents in asthma therapy and are the treatment of choice for acute asthma attacks. Despite the clinical importance of beta-agonists, only recently have we begun to understand the cellular and molecular mechanisms that regulate beta2AR responsiveness in ASM. Characterizing such mechanisms, and the context in which they occur, is of clinical relevance because the loss of Beta2AR responsiveness (desensitization) may contribute to a loss of therapeutic beta-agonist efficacy. Studies performed under our original grant identified numerous physiologically-relevant agents that promote Beta2AR desensitization in ASM, and identified roles of various protein kinases (G-protein-coupled receptor kinases (GRKs), protein kinase (PK) A, and PKC) and arrestins in mediating Beta2AR desensitization. Among the relevant agents shown to promote Beta2AR desensitization is the cytokine IL-1 Beta, which in conjunction with growth factors can desensitize Beta2ARs as well as PGE2 receptors (EPRs) through a mechanism involving induction of cyclooxygenase-2 (COX-2) and PGE2 synthesis and possibly other ill-defined mechanisms. Interestingly, both IL- 1Beta and growth factors also promote upregulated activity or sensitization of adenylyl cyclase (AC), the downstream effector of the BE2AR that is ultimately responsible for generating cAMP, the second messenger critical to transducing signals initiated by Beta2ARs and EPRs. The current proposal seeks to elucidate the mechanisms by which cytokines and growth factors promote both desensitization of the Beta2AR and sensitization of AC in ASM. Mechanisms of cytokine-mediated Beta2AR desensitization in ASM will be explored by analyzing the relative contributions of COX-2/PGE2 induction, PKA-mediated phosphorylation of the BE2AR, and altered expression or activity of GRKs and arrestins. Mechanisms of AC sensitization will be identified by assessing alterations in AC isoform expression, phosphorylation, or compartmentalization in cellular microdomains. Lastly, we will link altered GPCR signaling promoted by IL- 1Beta to alterations in ASM function by examining the regulation of ASM contractile state in tracheal rings derived from EP2R knockout mice. Experimental designs for these Aims will emphasize immunoprecipitation and cell fractionation strategies, ex vivo models of ASM function utilizing cells/tissues from EP2R knockout mice, and recently-development techniques for expression of recombinant signaling proteins in ASM cultures. These studies should uncover novel paradigms of Beta2AR and AC regulation and hopefully foster the development of more effective asthma therapies.